Despite recent successes, the limited number of pancreata available for transplantation will limit the utility of islet transplantation for the treatment of diabetes in the absence of new sources of islets. One possible source of islets is embryonic stem (ES) cells. ES cells are pluripotent cells that have the capacity to differentiate into cells from all three embryonic lineages. Recently, the differentiation of ES cells into islet-like structures was reported, although the insulin content of the islets was markedly reduced, thus limiting their capacity to correct hyperglycemia in diabetic mice. The hypothesis of the studies in this proposal is that the use of gene transfer to express PDX-1, a transcription factor important for both islet cell development and beta cell function, in ES cells will enhance islet differentiation and insulin secretory capacity. Initial studies will build upon the reported success of differentiating islets from ES cells by using an adenoviral vector that expresses PDX-1 (Ad-PDX-1) to infect partially differentiated ES cells with the goal of increasing the insulin secretory capacity of the islets. The resulting islet cells will be examined for expression of islet-specific genes, insulin secretion in response to glucose and other secretagogues and in vivo function, as assessed using islet transplantation into mice with streptozotocin-induced diabetes. A second series of studies will determine whether PDX-1 expression in undifferentiated ES cells will facilitate differentiation along an islet lineage upon differentiation of the ES cells in embryoid bodies (EBs). Following periods of differentiation, the EBs will be examined for expression of islet-specific genes and in vitro and in vivo function as described above. As environmental signals from surrounding cells have the capacity to promote ES cell differentiation along specific cell lineages, we will also determine whether differentiation of ES cells infected with Ad-PDX-1 into islet cells is promoted by exposure to the renal subcapsular space, which has been shown to promote differentiation of pancreatic epithelium from mouse embryos into islets. Thus, the proposed studies will address novel ways of promoting the differentiation of ES cells into islets with the ultimate goal of enhancing islet cell transplantation. [unreadable] [unreadable]